1. Field of the Invention
The present invention relates to improved compositions for the prevention and treatment of smallpox, and in particular to the use of compositions containing an antibody that binds to an epitope found on the MV form of the smallpox virus and an antibody that binds to an epitope found on the EV form of the smallpox virus. The invention relates to such compositions, especially to non-blood derived antibody compositions, such as chimeric or humanized antibodies, and to methods for their use in imparting passive immunity against smallpox infection to individuals at risk of smallpox virus infection or who exhibit smallpox.
2. Description of Related Art
“Smallpox” (also known by the Latin names Variola or Variola vera) is a disease caused by viruses (“smallpox viruses”) of the family poxyiridae, subfamily chordopoxyirinae. The lifecycle of poxviruses is complicated by having multiple infectious forms, with differing mechanisms of cell entry. It is a large virus, with a double stranded DNA genome of about 200 kilobases, making it more complicated than the smallest bacteria.
The infectious dose of variola virus is very small, possibly only a few virions. Respiratory infection is characterized by non-infectious incubation and prodromal periods that normally last 12-14 and 2-4 days, respectively (McClain, D. J. (1997) “Smallpox,” In: TEXTBOOK OF MILITARY MEDICINE (Zatjchuk R, ed.); Washington, D.C.: Office of the Surgeon General, Walter Reed Medical Center; pp. 539-559). This non-infectious period may represent the window of opportunity for post-exposure treatment by a neutralizing monoclonal antibody product. Variola travels from the upper to lower respiratory tract to regional lymph nodes where it replicates. Asymptomatic viremia occurs on about the third or fourth day after infection and variola then disseminates systemically to other lymphoid tissues, spleen, liver, bone marrow and lung. A second viremia begins at about the eighth day. The prodromal period, which begins about day 12, is characterized by high fever, malaise, backache and headache. At this time, the virus, contained in leukocytes, localizes in small blood vessels of the dermis and beneath the oral and pharyngeal mucosa and subsequently infects adjacent cells. The onset of the infectious state is characterized by the appearance of enanthem (oropharyngeal lesions) and soon followed by exanthem (skin lesions) on the face, hands and forearms. The lesions on mucous membranes shed infected epithelial cells and give rise to infectious oropharyngeal secretions, which are the most important means of virus transmission to contacts. Rash spreads centrally during the next week to the trunk and lesions progress from macules to papules to pustular vesicles. Lesions display a centrifugal distribution and generally remain in a synchronous stage of development in various segments of the body. Scab formation occurs 10-14 days after onset of rash and scab separation occurs during the following 14 days. Because virus can be isolated from scabs, patients are considered infectious until all scabs separate. Smallpox has little effect on the vital organs in the body. Death, which generally occurs during the second week of illness, is usually the result of the toxemia associated with circulating immune complexes and soluble variola antigens, which leads to disseminated intravascular coagulation, hypotension and cardiovascular collapse.
Person-to-person transmission of smallpox is initiated by the deposition of virus in the respiratory tract of the contact; the virus having originated from oropharyngeal secretions of a person with a rash. Epidemiological studies have implicated large-particle aerosol droplets, resuspension of virus particles, and droplet nuclei as possibly important in natural transmission of variola virus (Downie, A. W. et al. (1958) “The Antibody Response In Man Following Infection With Viruses Of The Pox Group. III. Antibody Response In Smallpox,” J. Hyg. (Lond) 56(4):479-487). The large-particle aerosol droplets are thought to infect the upper respiratory tract, where as the droplet nuclei may initiate infection in the lower respiratory tract. A bioterrorist release of variola or monkeypox viruses is likely to be by droplet nuclei, and thus the lower respiratory tract may be a site of infection. The intranasal and aerosol methods of infection mimic large-particle droplet and droplet nuclei modes of infection, respectively.
Smallpox virus infection is associated with very high morbidity and high overall mortality rates. The classic (or ordinary) form of variola major infection resulted in fatality rates of 30% for unvaccinated individuals and 3% for vaccinated individuals (McClain, D. J. (1997) “Smallpox,” In: TEXTBOOK OF MILITARY MEDICINE (Zatjchuk R, ed.); Washington, D.C.: Office of the Surgeon General, Walter Reed Medical Center; pp. 539-559). Approximately 2-5% of cases presented as flat-type smallpox, which was associated with severe systemic toxicity and fatality rates of 95% for unvaccinated individuals and 66% for vaccinated individuals. Fewer than 3% of cases presented as hemorrhagic-type smallpox, which was uniformly fatal; death occurred before typical pox lesions developed. Infection with variola minor, a less virulent variant of the variola virus, resulted in fatality rates of 1% in unvaccinated individuals. Patients who recovered from smallpox may be left with serious sequelae, including extensive scarring of the skin, hearing loss or blindness or, more rarely, other organ damage. Smallpox was highly contagious; infection invariably resulted in symptomatic disease. The aerosolized virus was capable of spreading over considerable distances, and of inducing infection even at low viral doses.
Smallpox was responsible for an estimated 300-500 million deaths in the 20th century alone. In light of the enormous medical and social significance of the disease, a concerted worldwide inoculation campaign was initiated to eradicate smallpox. In 1979, the World Health Organization certified that the campaign to eliminate naturally occurring smallpox had been successful (Geddes, A. M. (2006) “The History of Smallpox,” Clin. Dermatol. 24(3):152-157). Smallpox remains the sole human infectious disease to have been completely eradicated from nature (see, e.g., Bazin, H. (2000) THE ERADICATION OF SMALLPOX: EDWARD JENNER AND THE FIRST AND ONLY ERADICATION OF A HUMAN INFECTIOUS DISEASE; Academic Press, NY).
The occurrence of the 2001 U.S. Postal anthrax spore incident has demonstrated the possibility that biological agents might be used by terrorists against population centers. Because bioterrorism has moved from a theoretical risk to a reality, Governments have recognized the importance of taking steps to protect their populations from the consequences of biological and other unconventional terrorist attacks.
Of the organisms that might potentially be used as bioweapons, smallpox poses one of the most formidable risks to public health (Henderson, D. A. (1998) “Bioterrorism as a public health threat,” Emerg. Infect. Dis. 1998 July-September; 4(3):488-92; Wittek, R. (2006) “Vaccinia Immune Globulin Current Policies, Preparedness, And Product Safety And Efficacy,” Int. J. Infect. Dis. 10(3):193-201). As discussed above, the disease exhibits high morbidity, mortality and contagiousness even at low doses. Vaccinations against smallpox were stopped in light of the eradication of the naturally occurring disease. Thus, most people have not been vaccinated and among those who have been vaccinated, immunity to the virus is declining due to the passage of time. As a consequence, U.S. and world populations are now highly susceptible to smallpox. In light of the latency period associated with the disease, if virus was released in an unnoticeable manner—for example, by exploding a light bulb containing virus in a city subway system (Henderson, D. A. (1998) “Bioterrorism As A Public Health Threat,” Emerg. Infect. Dis. 1998 July-September; 4(3):488-492)—the resultant infections would be undetected for approximately 2 weeks and could thus easily be spread to numerous contacts. Significantly, the inexperience of current-day physicians with diagnosing smallpox would introduce additional delays in effecting treatment and thus would allow further spread to occur. As stated above, vaccination at the time of appearance of symptoms would be too late to provide effective protection. Accordingly, despite the eradication of naturally occurring smallpox, a need exists for modalities capable of protecting world populations from a smallpox bioweapons attack.
One approach for achieving such protection would be to re-initiate a vaccination regimen. First generation, conventional smallpox vaccines used live vaccinia virus prepared from calf lymph (e.g., Dryvax® (Wyeth Laboratories) or Lister Elstree (Kretzschmar, M. et al. (2006) “Frequency of Adverse Events after Vaccination with Different Vaccinia Strains,” PLoS Medicine 3(8):e272 doi:10.1371/journal.pmed.0030272). Although these vaccines were employed to eradicate smallpox worldwide, they cause a relatively high level of complications and are contraindicated in populations such as the immunosuppressed, pregnant females and the very young (Fulginiti, V. A. et al. (2003) “Smallpox Vaccination: A Review, Part II. Adverse Events,” Clin. Infect. Dis. 37(2):251-271). In addition, due to the emergence of myo/pericarditis in vaccinees in recent civilian and military vaccination programs, contraindications have been updated to include persons with cardiac disease or certain risk factors for cardiac disease (Centers for Disease Control and Prevention (2003) “Update: Cardiac-Related Events During The Civilian Smallpox Vaccination Program—United States, 2003,” Morbidity Mortality Weekly Rep. 52:492-496). Thus, live vaccinia virus vaccines prepared from calf lymph are no longer considered acceptable by current standards for human biologicals.
In the event of an imminent bioterrorist attack, vaccinia virus vaccine could be administered prophylactically. In case of exposure from a bioterrorist attack, post-exposure vaccination can be effective, particularly when utilized in a ring vaccination strategy (Kretzschmar, M. et al. (2004) “Ring Vaccination And Smallpox Control,” Emerg. Infect. Dis. 10(5):832-841). However, the window of opportunity for treatment of infected individuals is short—the vaccine must be administered within 4 days of first exposure to offer some protection against acquiring infection and significant protection against fatal outcome (Dixon, C. W. (1962) SMALLPOX (1st Ed.) London: Churchill Ltd.; Henderson, D. A. et al. (1999) “Smallpox As A Biological Weapon: Medical And Public Health Management. Working Group On Civilian Biodefense,” J. Amer. Med. Assn. 281(22):2127-2137). The ineffectiveness of vaccination at later times most likely reflects the inability to generate a robust antibody response within the 2 week period prior to the onset of disease. Studies with monkeypox virus in rhesus monkeys have established that neutralizing antibodies are the most critical arm of a protective immune response against orthopoxvirus infection (Edghill-Smith, Y. et al. (2005) “Smallpox Vaccine Does Not Protect Macaques With AIDS From A Lethal Monkeypox Virus Challenge,” J. Infect. Dis. 191(3):372-381; Edghill-Smith, Y. et al. (2005) “Smallpox Vaccine-Induced Antibodies Are Necessary And Sufficient For Protection Against Monkeypox Virus,” Nat. Med. 11(7):740-747).
Second generation vaccines are being developed in light of such deficiencies. ACAM2000™ (Acambis Inc.) is a live vaccinia virus vaccine derived from a Dryvax plaque and produced from large scale Vero cell bioreactors under serum-free conditions. ACAM2000™, however, has been reported to have a vaccination success rate, antibody response, and safety profile similar to that of Dryvax® (Artenstein, A. W. et al. (2005) “A Novel, Cell Culture-Derived Smallpox Vaccine In Vaccinia-Naive Adults,” Vaccine 23(25):3301-3309). ACAM2000® has been approved by the U.S. Food & Drug Administration for use in persons determined to be at high risk of smallpox infection.
MVA-BN® (Bavarian Nordic) (or IMVAMUNE® (Bavarian Nordic)) is a third generation smallpox vaccine candidate being developed for use in individuals, such as immunocompromised individuals, who are contraindicated for the conventional vaccine. MVA (modified vaccinia virus Ankara) is a highly attenuated virus, having been passaged more than 500 times in chicken embryo fibroblast cells. MVA is replication deficient in most mammalian cell lines and subcutaneous vaccination with MVA does not result in a “vaccine take” (pustule, scab, and scar) (Stittelaar, K. J. et al. (2005) “Modified Vaccinia Virus Ankara Protects Macaques Against Respiratory Challenge With Monkeypox Virus,” J. Virol. 79(12):7845-7851). Vaccinations with MVA-BN® are reported to be well-tolerated and to lead to seroconversion at high dose, indicating that the vaccine appears to be efficacious (see Vollmar, J. et al. (2006; Epub 2005 Nov. 28) “Safety And Immunogenicity Of IMVAMUNE, A Promising Candidate As A Third Generation Smallpox Vaccine,” Vaccine 24(12):2065-2070). Significantly, MVA-BN is administered by injection instead of by pricking the skin with a bifurcated needle, thus, unlike conventional vaccines it is not easy to distinguish vaccinated individuals from unvaccinated individuals. The vaccine's effectiveness in “at-risk” individuals has not yet been determined. Moreover, it is not known whether any of the second or third generation vaccines would be capable of eliciting protective immunity rapidly enough to protect populations in the event of a smallpox bioweapon attack.
Passive antibody products have been proposed as an alternative approach for defending against biological weapons. Such products may be used either as a prophylactic or as a therapeutic regime in the period immediately following bioweapon exposure (Casadevall, A. et al. (2004) “Passive Antibody Therapy for Infectious Diseases,” Nat. Rev. Microbiol. 2(9):695-703; Henderson, D. A. et al. (1999) “Smallpox As A Biological Weapon: Medical And Public Health Management. Working Group On Civilian Biodefense,” J. Amer. Med. Assn. 281(22):2127-2137).
Vaccinia-immune globulin (VIG) has been recommended for the treatment of serious complications of smallpox vaccination, although its therapeutic value has not been fully proven (Kempe, C. H. (1960) “Studies Smallpox And Complications Of Smallpox Vaccination,” Pediatrics 26:176-189; Kempe, C. H. et al. (1961) “The Use Of Vaccinia Hyperimmune Gamma-Globulin In The Prophylaxis Of Smallpox,” Bull World Health Organ. 25:41-48; Bray, M. et al. (2003) “Progressive Vaccinia,” Clin. Infect. Dis. 36(6):766-774; Thorne, C. D. et al. (2003) “Emergency Medicine Tools To Manage Smallpox (Vaccinia) Vaccination Complications: Clinical Practice Guideline And Policies And Procedures,” Ann. Emerg. Med. 42(5):665-680; Hopkins, R. J. et al. (2004) “Clinical Efficacy Of Intramuscular Vaccinia Immune Globulin: A Literature Review,” Clin. Infect. Dis. 39(6):819-826; U.S. CDC (2002) “Vaccinia Immune Globulin: Indications, Precautions & Contraindications,”). VIG is a pool of γ-globulin that was originally collected from convalescent patients, but is now collected from vaccinees (Kempe, C. H. et al. (1956) “Hyperimmune Vaccinal Gamma Globulin; Source, Evaluation, And Use In Prophylaxis And Therapy,” Pediatrics 18(2):177-188; Anderson, S. G. et al. (1970) “The International Standard For Anti-Smallpox Serum,” Bull. World Health Organ. 42(4):515-523). Because initial preparations of VIG contained a high proportion of aggregated protein, it could not be administered intravenously (IV), but was instead needed to be injected intramuscularly (IM). After certification of the eradication of smallpox, no international stockpile of VIG was constituted or maintained, and national stocks were not renewed as they aged and decreased in potency. However, the emergence of HIV has placed restrictions on the safe and effective use of smallpox vaccines and made the need for VIG, or an alternative immunotherapeutic preparation, an important consideration for outbreak control.
VIGIV is a new formulation of VIG that is suitable for IV administration; it is prepared from the plasma of vaccinated military personnel. VIGIV was approved by the FDA in 2005 (Shearer, J. D. et al. (2005) “Biological Activity Of An Intravenous Preparation Of Human Vaccinia Immune Globulin In Mouse Models Of Vaccinia Virus Infection,” Antimicrob. Agents Chemother. 49(7):2634-2641; Hopkins, R. J. et al. (2004) “Safety And Pharmacokinetic Evaluation Of Intravenous Vaccinia Immune Globulin In Healthy Volunteers,” Clin. Infect. Dis. 39(6):759-766). According to CDC estimates, 21-247 VIG-treatable complication will be encountered per million vaccinees. Thus, for a U.S. population of 302,000,000, a stockpile of 6,300-75,000 doses of VIG would be required.
In patients infected with smallpox, VIG has been suggested to suppress the secondary viremia and to ameliorate the disease (Kempe, C. H. et al. (1956) “Hyperimmune Vaccinal Gamma Globulin; Source, Evaluation, And Use In Prophylaxis And Therapy,” Pediatrics 18(2):177-188). In a murine model in which onset of disease occurs approximately 4 days following intranasal vaccinia virus infection, VIG was effective when administered prior to the appearance of disease (Law, M. et al. (2005) “An Investigation Of The Therapeutic Value Of Vaccinia-Immune IgG In A Mouse Pneumonia Model,” J. Gen. Virol. 86(Pt 4):991-1000). These data are consistent with the limited studies of the 1950's and 1960's that show that VIG appears to be effective in variola virus-infected human subjects if administered at or before the onset of disease (Kempe, C. H. et al. (1956) “Hyperimmune Vaccinal Gamma Globulin; Source, Evaluation, And Use In Prophylaxis And Therapy,” Pediatrics 18(2):177-188; Kempe, C. H. et al. (1961) “The Use Of Vaccinia Hyperimmune Gamma-Globulin In The Prophylaxis Of Smallpox,” Bull World Health Organ. 25:41-48). VIG contains relatively high titers of anti-A33, anti-B5 and anti-A27 antibodies, but is relatively deficient in anti-L1 antibodies (Lawrence, S. J. et al. (2007) “Antibody Responses To Vaccinia Membrane Proteins After Smallpox Vaccination,” J. Infect. Dis. 196(2):220-229). Unfortunately, blood-derived products, such as VIG and VIGIV, have inherent deficiencies, are variable in activity and are limited in supply.
Monkeypox is a disease caused by an orthopoxvirus (monkeypox virus) (Bricaire, F. et al. (2006) “Emerging Viral Diseases,” Bull. Acad. Natl. Med. 190(3):597-608, 609, 625-627; Heeney, J. L. (2006) “Zoonotic Viral Diseases And The Frontier Of Early Diagnosis, Control And Prevention,” J. Intern. Med. 260(5):399-408; Sale, T. A. et al. (2006) “Monkeypox: An Epidemiologic And Clinical Comparison Of African And US Disease,” J. Am. Acad. Dermatol. 55(3):478-481; Nalca, A. et al. (2005) “Reemergence Of Monkeypox: Prevalence, Diagnostics, And Countermeasures,” Clin. Infect. Dis. 41(12):1765-1771; Prichard, M. N. et al. (2005) “Orthopoxvirus Targets For The Development Of Antiviral Therapies,” Curr. Drug Targets Infect. Disord. 5(1):17-28; Ligon, B. L. (2004) “Monkeypox: a review of the history and emergence in the Western hemisphere,” Semin. Pediatr. Infect. Dis. 15(4):280-287; Abrahams, B. C. et al. (2004) “Anticipating Smallpox And Monkeypox Outbreaks: Complications Of The Smallpox Vaccine,” Neurologist 10(5):265-274; Di Giulio, D. B. et al. (2004) “Human Monkeypox: An Emerging Zoonosis,” Lancet Infect. Dis. 4(1):15-25 (Erratum; Lancet Infect. Dis. (2004) 4(4):251); Heymann, D. L. et al. (1998) “Re-Emergence Of Monkeypox In Africa: A Review Of The Past Six Years,” Brit. Med. Bull. 54(3):693-702; Cho, C. T. et al. (1973) “Monkeypox Virus,” Bacteriol. Rev. 37(1):1-18).
Cases of human monkeypox are increasing in Africa, and an outbreak even occurred in the U.S. in 2003 (Parker, S. et al. (2007) “Human Monkeypox: An Emerging Zoonotic Disease,” Future Microbiol. 2:17-34). Monkeypox presents as a smallpox-like rash preceded by a 10-14 day incubation period. A prodromal fever, malaise and severe lymphadenopathy are common; mortality is approximately 10%. Monkeypox may be transmitted from human-to-human. However, unlike variola virus, which has no nonhuman host, monkeypox virus is amplified and maintained in wild-animal populations, such as rodents and nonhuman primates. Because of the higher prevalence of immunocompromised individuals, particularly in Sub-Saharan Africa as a result of the spread of HIV infection, the impact of human vaccination as an effective control measure is declining. The increased frequency of human monkeypox infections, especially in immunocompromised individuals, might permit the monkeypox virus to evolve and maintain itself independently in human populations. Monkeypox virus could also be used as a bioweapon; it is readily available in Africa and its genome could be manipulated to increase its virulence, such as by inserting an IL-4 gene (Parker, S. et al. (2007) “Human Monkeypox: An Emerging Zoonotic Disease,” Future Microbiol. 2:17-34).
In sum, despite all prior advances, a need remains for compositions that could be used to provide a rapid and effective response to an orthopox virus (e.g., smallpox virus, monkeypox virus, etc.) bioweapon attack. The present invention, which provides a cocktail of neutralizing monoclonal antibodies is directed to this and related needs.